Candidate: The candidate, Dr. Kristian Swearingen, is an accomplished analytical chemist with multiple publications detailing the development of instrumentation for the quantification of proteins. He now wishes to engage in a period of mentored research in parasitology in order to achieve his long-term goal of becoming an independent researcher applying the tools of instrumental analysis to the problem of infectious disease. Environment: Dr. Swearingen will be mentored by Dr. Stefan Kappe of Seattle BioMed and Dr. Robert Moritz of the Institute for Systems Biology. Dr. Kappe is an expert in Plasmodium biology, while Dr. Moritz is an expert in mass spectrometry of proteins. Dr. Kappe and Dr. Moritz have an established collaboration that has produced publications describing the use of mass spectrometry to characterize protein expression in Plasmodium. The proposed research will be carried out in equal part at Seattle BioMed and the Institute for Systems Biology, which is the sponsoring institution for this award. There is an established inter-institutional partnership between the Institute for Systems Biology and Seattle BioMed that includes joint appointments and shared resources. Personnel from both organizations move freely between the two organizations (separated by less than one city block), and core facilities and equipment are shared. Dr. Swearingen will benefit greatly from the collaborative research environments and the state-of-the-art facilities available at both institutes. Research: The disease malaria is caused by parasites of the genus Plasmodium. Malaria sickens hundreds of millions of people annually, yet there are limited treatments and no licensed vaccine. Plasmodium sporozoites are introduced into the mammalian host in the saliva of mosquitoes, whereupon the parasites migrate to the liver and invade hepatocytes. Clinical onset of malaria does not occur until the parasites exit the liver as merozoites and invade erythrocytes, reproducing asexually. A subset of merozoites differentiates into gametocytes. Upon transmission to mosquitoes, gametocytes mature into gametes, reproduce sexually, and produce more sporozoites, completing the life cycle. Preventing sporozoite infection of host hepatocytes would prevent onset of disease. Preventing differentiation of merozoites into gametocytes and eventually gametes would prevent transmission to the mosquito vectors that spread the disease. Phosphorylation is a reversible post-translational modification of proteins that regulates cell function. Inhibiting phosphorylation with kinase inhibitors can prevent Plasmodium invasion and transmission, but few of the proteins involved in this regulatory network are known. I will identify phosphorylation events essential to sporozoite infection of hepatocytes and sexual differentiation of merozoites, and then I will identify the kinases responsible, revealing targets for new antimalarials. The first two aims of this proposal are to identify phosphorylation events that trigger infectivity and transmission, respectively. Protein expression and phosphorylation in Plasmodium parasites will be quantified by mass spectrometry. Sporozoites isolated from the mosquito mid-gut, which have very low infectivity, will be compared with infectious salivary gland sporozoites that are untreated, treated with albumin to mimic arrival in the blood stream and induce gliding motility, or treated with heparin to mimic arrival in the liver and induce invasion. Undifferentiated merozoites will be compared with gametocytes and gametes. The third aim will be validation of important phosphorylation events identified in Aims 1 and 2. Transgenic parasites will be generated with amino acid substitutions that mimic the constitutive presence or absence of phosphorylation on key proteins, and the effect on invasion of hepatocytes or transmission to mosquitoes will be assessed. Hepatocyte invasion will be measured in vitro by fluorescence aided cell sorting and in vivo by measuring the time to blood stage patency in infected mice. Transmission to mosquitoes will be measured by feeding mosquitoes with infected blood and dissecting to check for development of sporozoites. Finally, the kinases responsible for the critical phosphorylation events identified above will be identified. Purified bait proteins will be immobilized on beads an exposed to cell lysate. Chemical cross-linkers will be used to capture the kinase-substrate complex, and the kinases will be identified by mass spectrometry.